Pd-1 specific antibodies and uses thereof

ABSTRACT

One aspect of the present disclosure provides antibodies that can act as agonists of PD-1, thereby modulating immune responses regulated by PD-1. Another aspect of the disclosure provides compositions comprising PD-1 specific antibodies and their use in methods of down regulating the immune response. These methods can be practiced on any subject, including humans or animals. Anti-PD-1 antibodies disclosed herein may be used, in another aspect of the invention to detect PD-1 or its fragments in a biological sample. The amount of PD-1 detected may be correlated with the expression level of PD-1, and associated with the activation status of immune cells (e.g., activated T cells, B cells, and/or monocytes) in the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/062,559, filed on Mar. 7, 2011, now U.S. Pat. No. 8,927,697, which isthe U.S. national stage application of International Patent ApplicationNo. PCT/IB2009/006946, filed Sep. 14, 2009, which claims the benefit ofU.S. Provisional Patent Application No. 61/096,485, filed Sep. 12, 2008,now abandoned, the disclosures of which are hereby incorporated byreference in their entireties, including all figures, tables and aminoacid or nucleic acid sequences.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Sep. 10, 2009 and is 26 KB. The entire contents ofthe sequence listing is incorporated herein by reference in itsentirety.

SUMMARY OF THE INVENTION

One aspect of the present disclosure provides antibodies that can act asagonists of PD-1, thereby modulating immune responses regulated by PD-1.In one embodiment, the anti-PD-1 antibodies can be novel antigen-bindingfragments. Anti-PD-1 antibodies disclosed herein are able to bind tohuman PD-1 and agonize the activity of PD-1, thereby inhibiting thefunction of immune cells expressing PD-1. Exemplary antibodies for usein the context of this disclosure include, but are not limited tomonoclonal antibody produced by clone 10.

Another aspect of the disclosure provides compositions comprising PD-1specific antibodies and their use in methods of down regulating theimmune response. These methods can be practiced on any subject,including humans or animals. In particular embodiments, anti-PD-1antibodies are used to treat or prevent immune disorders by reducing theT cell response. Non-limiting examples of immune disorders that can betreated via the administration of PD-1 specific antibodies to a subjectinclude, but are not limited to, rheumatoid arthritis, multiplesclerosis, inflammatory bowel disease, Crohn's disease, systemic lupuserythematosus, type I diabetes, transplant rejection, graft-versus-hostdisease, hyperproliferative immune disorders, cancer, and infectiousdiseases. Some embodiments of this aspect of the invention may use twoPD-1 specific antibodies that bind to distinct, non-overlappingepitopes.

Anti-PD-1 antibodies disclosed herein may be used, in another aspect ofthe invention to detect PD-1 or its fragments in a biological sample.The amount of PD-1 detected may be correlated with the expression levelof PD-1, and associated with the activation status of immune cells(e.g., activated T cells, B cells, and/or monocytes) in the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Detection of antibody binding to human PD-1 transfectants. Cellswere transfected with constructs expressing the extracellular region ofhuman PD-1 (panels A and B) or mutants that affected the binding ofClone 19 (L16R; panel C) or Clone 10 (L103E; panel D) anti-PD-1antibodies. The cells were labelled with isotype control antibody (panelA) or with Clone 19 (panels B and C) or Clone 10 (panel D) antibody,followed by Alexa647 labelled secondary antibody. Transfected 293T cellsare eGFP-positive (x-axis). Antibody binding is shown on the y-axis.

FIG. 2: PD-1 epitope screen. The geometric means of the Alex647fluorescence levels for the GFP⁺ cells is given for each of the mutantsof PD-1, expressed as full length proteins in HEK 293T cells.

FIG. 3: Anti-PD-1 antibody epitopes. The epitopes were mapped byantibody binding analysis following expression of single-residue mutatedforms of PD-1 in HEK 293T cells. Mouse PD-1 residues equivalent to humanPD-1 residues that when mutated partially or fully block the binding ofClones 2, 10 and 19 antibody are highlighted in black on the mouse PD-1crystal structure (Zhang et al. Immunity 20, 337-47 (2004)).Mouse-equivalents of human PD-1 residues that have no effect on thebinding of the antibodies when mutated are coloured grey. The mutatedresidue numbers for the non-binding mutants are given alongside thestructure, for each antibody. Clone 2 and clone 10 antibodies appear tocompete with one another for binding to PD-1 based upon the results ofthis analysis.

FIGS. 4A-4B: IL-2 secretion induced by anti-PD-1 antibodies binding to ahPD-1/mCD3ζWT/mCD28 chimera. (FIG. 4A) A chimera consisting of theextracellular region of human PD-1 and the transmembrane and cytoplasmicregions of mouse (m) TCRζ and CD28 was expressed in DO11.10 cells. (FIG.4B) The cells were treated with immobilized anti-CD3 (KT3) or anti-PD-1antibodies and the amount of IL-2 released was measured.

FIG. 5: Quantification of monoclonal antibodies loaded ontotosyl-activated DYNALBEADS. Amount of anti-human CD3 OKT3 antibody usedper loading (10⁷ beads with 2.5 μg total antibody) is shown on thex-axis. Remaining amount of antibody was made up with Rabbit IgG oranti-PD-1 antibodies (Clone 19, Clone 10, Clone 2) to a total of 2.5 μg.The number of IgG1 (Rabbit IgG or anti-PD-1 antibodies; red bars) orIgG2a (OKT3; blue bars) molecules detected per bead is shown on they-axis. Green arrows indicate beads selected for use in the experimentsshown in FIG. 6. Values are averages of duplicates.

FIG. 6: Titration of anti-PD-1 antibodies coupled to tosyl-activatedDYNALBEADS. Bulk preparations of PBL were incubated with beadscontaining anti-CD3 and increasing amounts of anti-PD-1 antibodies(Clone 19 or Clone 10). Amount of anti-PD-1 antibody loaded per 10⁷beads (in a total of 2.5 μg mAb per 10⁷ beads) is shown on the x-axis.Proliferation (y-axis) was measured by CFSE dilution at day 5. Barsrepresent means of triplicates±SD.

FIG. 7: Stimulation of a PD-1/mCD28 chimera-expressing DO11.10 cell linewith titrations of two anti-PD-1 antibodies. DO11.10 cells expressing aPD-1/mCD28 chimera were incubated with titrations of anti-PD-1 Clone 19antibody (from 100 μg/ml to 0 μg/ml) and anti-PD-1 Clone 10 antibody(from 100 μg/ml to 0 μg/ml). Cells were then incubated in donkeyanti-mouse IgG antibody coated (500 μg/ml) 96 well plates for 48 hoursbefore tissue culture supernatant was assayed for IL-2 by ELISA.

FIG. 8: Activation of T cells with beads quantified for Ig content. PBLwere depleted of monocytes by plastic adherence (bulk PBL). The amountof anti-CD3 (OKT3) and anti-PD-1 antibodies (Clone 19 or Clone 10) wasquantified and is shown in the table (left) expressed as number ofmolecules per bead. Proliferation (y-axis) was measured by CFSE dilutionat day 5. Bars are duplicates±SD.

FIG. 9: Explanation for differential signaling by the two antibodies.Clone 19 induces stronger signaling by a hPD-1/mCD3ζWT/mCD28 chimerathan Clone 10. PD-1 has ITIM (inhibitory, blue) and ITSM (activating,red) tyrosine-based signaling motifs. It is suggested that, in vitro,Clone 19 triggers the phosphorylation of both motifs whereas Clone 10ligation results in phosphorylation of the inhibitory motif only,leading to more potent inhibitory signaling.

FIGS. 10A-10B: IL-2 secretion induced by anti-PD-1 antibodies binding toa hPD-1/mCD28 chimera. (FIG. 10A) A chimera consisting of theextracellular region of human PD-1 and the transmembrane and cytoplasmicregions of mouse CD28 was expressed in DO11.10 cells. (FIG. 10B) Thecells were treated with immobilized anti-CD3 (KT3) or anti-PD-1antibodies and the amount of IL-2 released was measured.

FIGS. 11A-11C: Strong signaling induced by a pair of antibodies bindingto a monomeric signaling protein. (FIG. 11A) Antibodies, which arebivalent, cause strong signaling by homodimeric receptors because theyare able to generate a high local density of signaling domains. (FIG.11B) In contrast, antibodies are only able to recruit pairs of monomericreceptors, such as PD-1, leading to much less intense signaling. (FIG.11C) By using antibodies that bind to two non-overlapping epitopes,higher densities of monomeric signaling receptors can be generated,giving much more potent signaling.

FIG. 12: Dissociation rates for Clone 2, 10 and 19 antibodies determinedby surface plasmon resonance-based analysis. The three antibodies and anegative control (OX-7) were bound indirectly to the biosensor surface,i.e. via a covalently coupled rabbit anti-mouse Fc antibody. Monomericsoluble human PD-1 was then injected to saturating levels over theimmobilized antibodies in the buffer 10 mM Hepes, 150 mM NaCl pH 7.4.Following injection of the soluble PD-1, the buffer only was injected,allowing dissociation of the bound soluble PD-1 from each of theantibodies simultaneously. Dissociation rates were fitted using Originv.5.0 software (MicroCal Software Inc, Northampton, Mass.) aftersubtraction of the dissociation rate for OX-7 dissociating from theanti-mouse Fc antibody.

FIG. 13: Inhibition of CD4⁺ T cell proliferation by anti-PD-1antibodies. CD4⁺ T cells were purified from human PBL by negativeselection and cultured with Dynalbeads coated with anti-CD3 plus control(BSA or MOPC21) or Clone 10 antibody. Proliferation (y-axis) wasmeasured by ³H-thymidine incorporation at day 6. Bars represent the % ofmaximal response (anti-CD3/BSA) and are the mean+/−S.E.M. of 4 differentdonor cultures.

DETAILED DESCRIPTION

The term “antibody”, as used in this disclosure, refers to animmunoglobulin or a fragment or a derivative thereof, and encompassesany polypeptide comprising an antigen-binding site, regardless ofwhether it is produced in vitro or in vivo. Thus, an antibody includes,but is not limited to, polyclonal, monoclonal, monospecific,polyspecific, bispecific, humanized, single-chain, chimeric, synthetic,recombinant, hybrid, mutated, and grafted antibodies.

The term “antibody fragment” or “an antigen binding fragment” includesantibody fragments such as Fab, F(ab′)₂, Fv, scFv, Ed, dab, and otherantibody fragments that retain antigen-binding function, i.e., theability to bind PD-1 specifically and/or that are produced from amonoclonal antibody disclosed herein. These fragments comprise anantigen-binding domain and can also, in some embodiments, agonize thefunction of PD-1. Antibodies disclosed herein, and fragments thereof,include those antibodies having altered glycosylation patterns whencompared to the parent antibody (e.g., the antibody produced by clone 10and/or clone 19).

As discussed above, the PD-1 antibodies disclosed herein are able toantagonize the activity and/or proliferation of lymphocytes by agonizingPD-1. The term “antagonize the activity” relates to a decrease (orreduction) in lymphocyte proliferation or activity that is at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. The term“antagonize” may be used interchangeably with the terms “inhibitory” and“inhibit”. PD-1-mediated activity can be determined quantitatively usingT cell proliferation assays as described herein.

The terms “therapeutically effective”, “therapeutically effectiveamount”, “effective amount” or “in an amount effective” refers to adosage or amount of the disclosed antibodies that is sufficient toagonize the activity of PD-1 and provide for the amelioration ofsymptoms in a subject or to achieve a desired biological response, e.g.,decreased T cell activity, etc.

The term “isolated” refers to a molecule that is substantially free ofits natural environment. For instance, an isolated antibody issubstantially free of cellular material or other proteins from the cell(e.g., hybridoma) or other source from which it is derived. The termisolated also refers to preparations where the isolated protein issufficiently pure to be administered as a pharmaceutical composition, orat least 70-80% (w/w) pure, at least 80-90% (w/w) pure, 90-95% pure; orat least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.

One aspect of the present disclosure provides antibodies that can act asagonists of PD-1, thereby modulating immune responses regulated by PD-1.In one embodiment, the anti-PD-1 antibodies can be novel antigen-bindingfragments. Anti-PD-1 antibodies disclosed herein are able to bind toincluding human PD-1 and agonize PD-1, thereby inhibiting the functionof immune cells expressing PD-1. In some embodiments, the immune cellsare activated lymphocytes, such as T-cells, B-cells and/or monocytesexpressing PD-1. Exemplary antibodies for use in the context of thisdisclosure include, but are not limited to monoclonal antibodiesproduced by clone 10. Some embodiments of this aspect of the inventionmay use two PD-1 specific antibodies that bind to distinct,non-overlapping epitopes. Other embodiments provide for antibodies thatcompete with one another for binding to an epitope present on PD-1(e.g., Clone 10 and Clone 2).

Anti-PD1 antibodies described herein can be linked to anothermolecule/moiety. Non-limiting examples include another peptide orprotein (albumin, another antibody, etc.), toxins, radioisotopes,cytotoxic agents or cytostatic agents. The term “link” or “linked”relates to the chemical cross-linking or covalent attachment of anothermolecule/moiety by recombinant methods. Antibodies disclosed herein mayalso be linked to one or more nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes (see, forexample, U.S. Pat. Nos. 4,791,192; 4,766,106; 4,670,417; 4,640,835;4,609,546; 4,496,689; 4,495,285; 4,301,144; and 4,179,337, which areeach hereby incorporated by reference in their entireties).

The antibodies may also be tagged with a detectable, or functional,label. Detectable labels include radiolabels such as ⁹⁹Tc, which mayalso be attached to antibodies using conventional chemistry. Detectablelabels also include enzyme labels such as horseradish peroxidase oralkaline phosphatase. Other types of detectable labels include chemicalmoieties such as biotin, which may be detected via binding to a specificcognate detectable moiety, e.g., labeled avidin.

Another aspect of the invention provides for the use of antibodiesdisclosed herein for isolating PD-1 or PD-1-expressing cells. Yetanother aspect of the invention provides methods of inducing toleranceto a specific antigen. For example, tolerance can be induced byco-administration of antigen and an anti-PD-1 antibody disclosed herein.Still other aspects of the invention relate to reducing immune responsesmediated by activated lymphocytes in a subject comprising theadministration of anti-PD-1 antibodies disclosed herein. Another aspectof the invention provides for the use of the disclosed anti-PD-1antibodies for agonizing PD-1 and down regulating immune responses (orin some cases inhibiting or reducing the proliferation of activatedlymphocytes). In particular embodiments, the immune response isTcR/CD28-mediated. As discussed herein, allergies, rheumatoid arthritis,type I diabetes mellitus, multiple sclerosis, inflammatory boweldisease, Crohn's disease, systemic lupus erythematosus, tissue, skin andorgan transplant rejection or graft-versus-host disease (GVHD) can betreated via the administration of anti-PD-1 antibodies. Some embodimentsof this aspect of the invention may use two PD-1 specific antibodiesthat bind to distinct, non-overlapping epitopes.

Another aspect of the disclosure provides compositions comprising PD-1specific antibodies and their use in methods of down regulating theimmune response (or reducing the proliferation of activated T-cells,B-cells or mononuclear cells). These methods can be practiced on anysubject, including humans or animals. In particular embodiments,anti-PD-1 antibodies are used to treat or prevent immune disorders byreducing the T cell response. Non-limiting examples of immune disordersthat can be treated via the administration of PD-1 specific antibodiesto a subject include, but are not limited to, rheumatoid arthritis,multiple sclerosis, inflammatory bowel disease, Crohn's disease,systemic lupus erythematosus, type I diabetes, transplant rejection,graft-versus-host disease, hyperproliferative immune disorders, cancer,and infectious diseases. Yet other aspects of the invention provide forinhibiting or reducing lymphocyte (T-cell, B-cell and/or monocyte)activity in inflammatory lesions. Some embodiments of this aspect of theinvention may use two PD-1 specific antibodies that bind to distinct,non-overlapping epitopes (such antibodies can be affinity matched toprovide a desired activity in vivo (e.g., Clone 19 and Clone 2)).

As illustrated in FIG. 12, the antibody produced by clone 10 has arelatively low affinity for PD-1. Such low affinity antibodies can beused in a manner similar to ligands of PD-1. For example, the Clone 10antibody has a very fast off-rate (similar to that for one of the nativeligands for PD-1 (i.e., PD-L2)). A fast off-rate gives good signaling byClone 10 in vitro because it may allow for the “serial engagement” ofmultiple PD-1 molecules. Thus, antibodies such as those produced byclone 10 can be used to engage numerous PD-1 molecules and causeinhibitory signaling.

Anti-PD-1 antibodies disclosed herein may be used, in another aspect ofthe invention to detect PD-1 or its fragments in a biological sample.The amount of PD-1 detected may be correlated with the expression levelof PD-1, and associated with the activation status of immune cells(e.g., activated T cells, B cells, and/or monocytes) in the subject.

Another aspect of the invention provides anti-PD-1 specific monoclonalantibodies having modified binding affinity. One embodiment provides formodifying the binding affinity such that the antibody has a low affinityfor PD-1 (e.g., the antibody has a dissociation rate of between 0.1sec⁻¹ and 0.5 sec⁻¹ or less than 0.90 sec⁻¹). Particular embodimentsprovided antibodies having off rates of 0.10 sec⁻¹, 0.15 sec⁻¹, 0.20sec⁻¹, 0.25 sec⁻¹, 0.30 sec⁻¹, 0.35 sec⁻¹, 0.40 sec⁻¹, 0.45 sec⁻¹ or0.50 sec⁻¹ or for antibodies having dissociation rates ranging from 0.04sec⁻¹ to 2.0 sec⁻¹ (e.g., 0.04 sec⁻¹, 0.05 sec⁻¹, 0.06 sec⁻¹, 0.07sec⁻¹, 0.08 sec⁻¹, 0.09 sec⁻¹, 0.10 sec⁻¹, 0.15 sec⁻¹, 0.20 sec⁻¹, 0.25sec⁻¹, 0.30 sec⁻¹, 0.35 sec⁻¹, 0.40 sec⁻¹, 0.45 sec⁻¹, 0.50 sec⁻¹, 0.55sec⁻¹, 0.60 sec⁻¹, 0.65 sec⁻¹, 0.70 sec⁻¹, 0.75 sec⁻¹, 0.80 sec⁻¹, 0.85sec⁻¹, 0.90 sec⁻¹, 0.95 sec⁻¹, 1.0 sec⁻¹, 1.10 sec⁻¹, 1.20 sec⁻¹, 1.30sec⁻¹, 1.40 sec⁻¹, 1.50 sec⁻¹, 1.60 sec⁻¹, 1.70 sec⁻¹, 1.80 sec⁻¹, 1.90sec⁻¹, or 2.00 sec⁻¹). Antibodies having such binding affinities can bemodified in any suitable process.

Thus, the binding affinity of the antibodies (such as those produced byclone 2, clone 10 or clone 19) can be increased or decreased via variousmethods known in the art. For example, binding characteristics can bemodified by direct mutation, methods of affinity maturation, phagedisplay, or chain shuffling within the nucleic acids encoding theantibody molecules. Individual residues or combinations of residues canbe randomized so that in a population of otherwise identical antigenbinding sites, all twenty amino acids are found at particular positionsand binding characteristics/affinities can also be modified by methodsof affinity maturation. (See, e.g., Yang et al. (1995) J. Mol. Biol.254, 392-403; Hawkins et al. (1992) J. Mol. Bio. 226, 889-896; or Low etal. (1996) J. Mol. Biol. 250, 359-368 (each of which is herebyincorporated by reference in its entirety, particularly with respect tomethods of increasing or decreasing the binding affinity ofantibodies)). Methods known in the art include, for example, Marks etal. BioTechnology, 10, 779-783 (1992), which describes affinitymaturation by VH and VL domain shuffling; random mutagenesis of CDRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91, 3809-3813 (1994); Schier et al. Gene, 169, 147-155 (1995);Yelton et al. J. Immunol., 155, 1994-2004 (1995); Jackson et al. J.Immunol., 154, 3310-9 (1995); and Hawkins et al. J. Mol. Biol., 226,889-896 (1992).

Strategies for antibody optimization are sometimes carried out usingrandom mutagenesis. In these cases positions are chosen randomly, oramino acid changes are made using simplistic rules. For example allresidues may be mutated to alanine, referred to as alanine-scanning. WO9523813 (which is hereby incorporated by reference in its entirety)teaches in vitro methods of altering antibody affinities utilizingalanine-scanning mutagenesis. Alanine-scanning mutagenesis can also beused, for example, to map the antigen binding residues of an antibody(Kelley et al. Biochemistry 32, 6828-6835 (1993); Vajdos et al. J. Mol.Biol. 320, 415-428 (2002)). Sequence-based methods of affinitymaturation (see, U.S. Pat. Application No. 2003/022240 A1 and U.S. Pat.No. 2002/177170A1, both hereby incorporated by reference in theirentireties) may also be used to increase or decrease the bindingaffinities of antibodies. Finally, the binding affinities of antibodiesin which the binding affinity has been altered can be determined usingmethods as disclosed herein (for example, dissociation rates formodified antibodies can be determined by surface plasmon resonance-basedanalysis as described for FIG. 12). T cells can be activated by anyT-cell activating compound. As discussed in the examples, one suchT-cell-activating compound is an anti-CD3 antibody, which binds TcR.Activating anti-CD3 antibodies are known in the art (see, for example,U.S. Pat. Nos. 6,405,696 and 5,316,763 [each of which is herebyincorporated by reference in its entirety]). The ratio between theactivating TcR signal and negative PD-1 signal is determinedexperimentally using conventional procedures known in the art or asdescribed in the Examples.

The antibodies or antibody compositions of the present invention areadministered in therapeutically effective amounts. Generally, atherapeutically effective amount may vary with the subject's age,condition, and sex, as well as the severity of the medical condition ofthe subject. A therapeutically effective amount of antibody ranges fromabout 0.001 to about 25 mg/kg body weight, preferably from about 0.01 toabout 25 mg/kg body weight, from about 0.1 to about 20 mg/kg bodyweight, or from about 1 to about 10 mg/kg. The dosage may be adjusted,as necessary, to suit observed effects of the treatment. The appropriatedose is chosen based on clinical indications by a treating physician.

In another aspect, the antibodies of the invention can be used as atargeting agent for delivery of another therapeutic or a cytotoxic agent(e.g., a toxin) to a cell expressing PD-1. The method includesadministering an anti-PD-1 antibody coupled to a therapeutic or acytotoxic agent or under conditions that allow binding of the antibodyto PD-1 expressed on the cell surface.

Still other aspects of the invention provide for the use of thedisclosed antibodies for detecting the presence of PD-1 in biologicalsamples. The amount of PD-1 detected may be correlated with theexpression level of PD-1, which, in turn, is correlated with theactivation status of immune cells (e.g., activated T cells, B cells, andmonocytes) in the subject.

The subject invention also provides methods of binding an antibody to aPD-1 polypeptide comprising contacting a sample that may contain PD-1 orcells expressing PD-1 with an antibody under conditions that allow forthe formation of an antibody-antigen complex. These methods can furthercomprise the step of detecting the formation of said antibody-antigencomplex. The complex can be detected using any means known in the art(e.g., fluorescence activated cell sorting, radioimmunoassays, orchromogenic assays).

Another aspect of the disclosure provides compositions comprisinganti-PD-1 antibodies. These compositions can be formulated according toknown methods for preparing pharmaceutically useful compositions.Formulations are described in a number of sources which are well knownand readily available to those skilled in the art. For example,Remington's Pharmaceutical Science (Martin E. W., Easton Pa., MackPublishing Company, 19^(th) ed., 1995) describes formulations which canbe used in connection with the subject invention. Formulations suitablefor administration include, for example, aqueous sterile injectionsolutions, which may contain antioxidants, buffers, bacteriostats, andsolutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and nonaqueous sterile suspensions whichmay include suspending agents and thickening agents. The formulationsmay be presented in unit-dose or multi-dose containers, for examplesealed ampoules and vials, and may be stored in a freeze dried(lyophilized) condition requiring only the condition of the sterileliquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, theformulations of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

Another aspect of the invention provides nucleic acids encoding PD-1specific antibodies disclosed herein. For example, the nucleic acidsencoding the antibody secreted by clone 10 or clone 2 can be isolatedaccording to methods known to those skilled in the art. Yet anotheraspect of the invention provides vectors and transformed host cellscomprising a nucleic acid encoding a PD-1 specific antibody as secretedby clone 10 or clone 2. As would be apparent to those skilled in theart, constant regions of the murine antibodies disclosed herein can besubstituted with human constant regions to form chimeric antibodiescomprising murine variable regions and human constant regions. Someembodiments provide for the substitution of heavy chain constant regionson the disclosed antibodies that provide for higher Fc receptor bindingby the antibodies (e.g., human IgG1, IgG3, and murine IgG2a isotypes,all of which bind Fc receptors strongly, can be grafted onto variableregions of the disclosed antibodies without affecting bindingspecificity). Alternatively, CDRs from the murine antibodies disclosedherein can be isolated and grafted into human framework regions to formhumanized antibodies. Finally, methods of producing the disclosed PD-1specific antibodies (including methods of producing the aforementionedhumanized and chimeric antibodies) are also provided by the subjectinvention.

The hybridomas disclosed herein were deposited on Sep. 9, 2008 withEuropean Collection of Cell Cultures (ECACC), Centre For EmergencyPreparedness and Response, The Health Protection Agency, Porton Down,Salisbury, Wiltshire, SP4 0JG United Kingdom. The accession numbers forthe hybridomas are as follows:

Clone 2: 08090903; Clone 10: 08090902; and Clone 19: 08090901.

As discussed above, antibodies disclosed herein can be a full-lengthmurine, human, humanized, or chimeric antibody; or a fragment orderivative thereof. In one embodiment, the antibody binds the same, orsubstantially the same, epitope as clone 10 or clone 2 or by amonoclonal antibody comprising: a) SEQ ID NO: 10 and SEQ ID NO: 8; or b)SEQ ID NO: 6 and SEQ ID NO: 2. In another embodiment, the antibody,including a fragment or derivative thereof, comprises the same orsubstantially identical VH and/or Vk regions as clone 10 (SEQ ID NOs: 10and 8) or clone 2 (SEQ ID NOs: 6 and 2).

In another embodiment, the antibody, including a fragment or derivativethereof, comprises the same or substantially identical CDR1, CDR2 andCDR3 regions as those found in the Vk and VH sequences of clone 10 orclone 2. In one embodiment, the antibody comprises: a) SEQ ID NO: 10 andSEQ ID NO: 8; or b) SEQ ID NO: 6 and SEQ ID NO: 2, as well as thesequence for murine IgG1 constant heavy chain region (GenBank accessionNo. D78344, hereby incorporated by reference in its entirety) and thesequence for murine IgG1 constant light chain region (GenBank accessionNo. V00807, hereby specifically incorporated by reference in itsentirety). Other aspects of the invention provide nucleotide sequencesencoding the disclosed antibodies, expression vectors comprising suchsequences, host cells comprising such vectors, and methods of producingsuch antibodies from such host cells.

Fragments and derivatives of antibodies of this invention can beproduced by techniques that are known in the art. “Immunoreactivefragments” comprise a portion of the intact antibody, generally theantigen binding site or variable region. Examples of antibody fragmentsinclude Fab, Fab′, Fab′-SH, F(ab′)₂, and Fv fragments; diabodies; anyantibody fragment that is a polypeptide having a primary structureconsisting of one uninterrupted sequence of contiguous amino acidresidues (referred to herein as a “single-chain antibody fragment” or“single chain polypeptide”), including without limitation (1)single-chain Fv (scFv) molecules (2) single chain polypeptidescontaining only one light chain variable domain, or a fragment thereofthat contains the three CDRs of the light chain variable domain, withoutan associated heavy chain moiety and (3) single chain polypeptidescontaining only one heavy chain variable region, or a fragment thereofcontaining the three CDRs of the heavy chain variable region, without anassociated light chain moiety; and multispecific antibodies formed fromantibody fragments. For instance, Fab or F(ab′)₂ fragments may beproduced by protease digestion of the isolated antibodies, according toconventional techniques. Alternatively, the DNA of a hybridoma producingan antibody of this invention may be modified so as to encode for afragment of this invention. The modified DNA is then inserted into anexpression vector and used to transform or transfect an appropriatecell, which then expresses the desired fragment.

In an alternate embodiment, the DNA of a hybridoma producing an antibodyof this invention can be modified prior to insertion into an expressionvector, for example, by substituting the coding sequence for humanheavy- and light-chain constant domains in place of the homologousnon-human sequences (e.g., Morrison et al., Proc. Natl. Acad. Sci.U.S.A., 81, pp. 6851 (1984)), or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of theoriginal antibody. Typically, such non-immunoglobulin polypeptides aresubstituted for the constant domains of an antibody of the invention.Thus, the antibodies of the present invention may also be made into“chimeric” antibodies (immunoglobulins) in which a portion of the heavyand/or light chain is identical with or homologous to correspondingsequences in the original antibody, while the remainder of the chain(s)is identical with or homologous to corresponding sequences in antibodiesderived from another species or belonging to another antibody class orsubclass, as well as fragments of such antibodies, so long as theyexhibit the desired biological activity (Cabilly et al., supra; Morrisonet al., Proc. Natl. Acad. Sci. U.S.A., 81, pp. 6851 (1984)).

In an exemplary embodiment, a chimeric recombinant mAb from clone 10 orclone 2 VH and Vk sequences, or a derivative or variant thereof, isproduced. Nucleic acid sequences encoding the clone 10 or clone 2 VH andVk sequences (SEQ ID NOs: 10 and 8 or SEQ ID NOs: 6 and 2, respectively)are cloned into a commercially available or otherwise known eukaryoticexpression vector containing the light and heavy chain constant regionsfor a human or non-human antibody, using standard techniques. Oneexample of a commercially available vector is pASK84, available from theATCC (American Type Culture Collection, catalog number 87094). CHOcells, or other mammalian cell lines are then transfected with thevectors by standard methods, as described for example in “MolecularCloning”, Sambrook et al. The result is transfected cell lines thatstably express and secrete the antibody molecule of interest, such as achimeric version of clone 10 or clone 2 comprising its original VH andVk regions and the constant regions from a human mAb. The entire cDNAsequences encoding the constant regions of human IgG can be found in thefollowing GenBank entries, each of which incorporated by reference inits entirety: Human IgG1 constant heavy chain region: GenBank accession#: J00228; Human IgG2 constant heavy chain region: GenBank accession #:J00230; Human IgG3 constant heavy chain region: GenBank accession #:X04646; Human IgG4 constant heavy chain region: GenBank accession #:K01316; and Human kappa light chain constant region: GenBank accession#: J00241.

Alternatively, VH and Vk regions of clone 10 or clone 2, or mutants orderivatives thereof, can be cloned into vectors encoding truncatedconstant regions in order to express antibody fragments (e.g., Fabfragments). Isotype-switching of antibody can be made according tosimilar principles. For example, an antibody with the exact samespecificity as clone 10 or clone 2 but of a different isotype can beobtained by sub-cloning the cDNA encoding Vk and VH sequences intoplasmids containing cDNA encoding human kappa light chain constantregions and a human heavy constant chain region selected from IgG1 orIgG2 or IgG3 or IgG4 constant heavy chain regions. Thus, an antibody asgenerated can possess any isotype and the antibody can then be isotypeswitched using conventional techniques in the art. Such techniquesinclude the use of direct recombinant techniques (see, e.g., U.S. Pat.No. 4,816,397), cell-cell fusion techniques (see e.g., U.S. Pat. No.5,916,771), and other suitable techniques known in the art. Accordingly,the effector function of antibodies provided by the invention may be“changed” with respect to the isotype of a parent antibody by isotypeswitching to, e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgMantibody for various therapeutic or other uses.

According to another embodiment, the antibody of this invention ishumanized. “Humanized” forms of antibodies according to this inventionare specific chimeric immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′, F(ab′) 2, or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from the murine immunoglobulin. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of the original antibody(donor antibody) while maintaining the desired specificity, affinity,and capacity of the original antibody. In some instances, Fv frameworkresidues of the human immunoglobulin may be replaced by correspondingnon-human residues. Furthermore, humanized antibodies can compriseresidues that are not found in either the recipient antibody or in theimported CDR or framework sequences. These modifications are made tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of the original antibody and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. For further details see Jones et al.,Nature, 321, pp. 522 (1986); Reichmann et al., Nature, 332, pp. 323(1988); and Presta, Curr. Op. Struct. Biol., 2, pp. 593 (1992).Accordingly, humanized versions of clone 10 or clone 2 antibodiescomprising the VH and Vk CDR regions of clone 10 or clone 2 and constantand framework regions from a human mAb can be made, using known constantand framework human mAb sequences and established techniques in the art,as described herein. For any humanized antibody incorporating the clone2 VH CDR1 domain, the domain can contain SEQ ID NO: 18 or amino acids6-10 of SEQ ID NO: 18. For any humanized antibody incorporating theclone 10 VH CDR1 domain, the domain can contain SEQ ID NO: 24 or aminoacids 6-11 of SEQ ID NO: 24.

Methods for humanizing the antibodies of this invention are well knownin the art. Generally, a humanized antibody according to the presentinvention has one or more amino acid residues introduced into it fromthe original antibody. These murine or other non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature, 321, pp. 522 (1986); Riechmann et al., Nature, 332, pp. 323(1988); Verhoeyen et al., Science, 239, pp. 1534 (1988)). Accordingly,such “humanized” antibodies are chimeric antibodies (Cabilly et al.,U.S. Pat. No. 4,816,567), wherein substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom the original antibody. In practice, humanized antibodies accordingto this invention are typically human antibodies in which some CDRresidues and possibly some FR residues are substituted by residues fromanalogous sites in the original antibody.

EXAMPLES Example 1 Methods for Generation of Anti-Pd-1 Antibodies 1.1Myeloma Cell Line

For fusion the myeloma cell line SP2/0-Ag14 from the German Collectionof Microorganisms and Cell Cultures (DSMZ GmbH, Braunschweig) was used.This cell line is a hybrid between BALB/c spleen cells and the myelomacell line P3×63Ag8. The cells have been described as not synthesizing orsecreting immunoglobulin chains, being resistant to 8-azaguanine at 20μg/ml, and not growing in HAT (Hypoxanthine, Aminopterin, Thymidine)medium. The SP2/0 cells are routinely maintained in tissue cultureflasks in standard growth medium (with 10% FCS). A new aliquot of frozenSP2/0 cells was used after a period of 2 weeks in order to avoid theimplementation of HGPRT-positive revertants. The myeloma cells wereshown to be negative in all mycoplasma tests.

1.2 Antigens for Immunization and Screening

The recombinant protein PD-1Fc was prepared using the methods describedfor the production of CD28Fc (Evans et al. Nat Immunol. 6, 271-9 (2005))and concentrated to 5.1 mg/ml in 0.01 M HEPES, 150 mM NaCl, pH 7.4.SDS-PAGE analysis of the antigen run under reducing and non-reducingconditions established the purity of the protein to be >95%.

1.3 Immunization

Five mice (about 8 weeks old) were immunized via the intraperitonealcavity using an immunization protocol over 60 days. For immunization analum precipitate of the immunogen was prepared. The alum precipitate wasfreshly prepared for each boost. The mice were immunized with 50 μgprotein and boosted with 25 μg protein. Three mice were used for fusion.

1.4 General Handling of Cells

Cells were handled under sterile conditions using a laminar air-flowsystem, sterile materials and sterile solutions. Cells were incubated at37° C. in a humid atmosphere containing 5% carbon dioxide. Forcultivation of the hybridoma cells a complete growth medium (CGM)containing DMEM with supplements 2-mercaptoethanol, L-Glutamine,GlutaMax, HT, non essential amino acids, sodium pyruvate,antibiotics/antimycotic solution (in concentrations recommended by thesupplier) and FCS at different concentrations (10%, 15% or 20%) wasused.

1.5 Preparation of Spleen Cells and Cell Fusions

After asphyxiation of the three immunized mice in CO₂ spleens wereaseptically removed. A single cell suspension of pooled spleens wasprepared. The spleen cells and the myeloma cells were washed severaltimes with DMEM and fused twice in the presence of 1 ml 50% (w/v) PEG3550 (ratio spleen cells to SP2/0 2.5:1 and 2.4:1). The hybridomasproduced were resuspended in CGM containing 20% FCS and aminopterin (HATmedium). The cell suspension (140 Cl/well) of each fusion was plated outinto eight 96-well tissue culture flat-bottom plates (Corning-Costar)containing 140 Cl/well peritoneal exudate cells as feeder cells in CGMwith 20% FCS. The plates were incubated for 10 days. During this periodcells were fed two times with HAT medium. An aliquot of the spleen cellpreparation (about 8×10⁶ spleen cells) was cultivated 10 days in a wellof a 24-well plate and the cell culture supernatant served as positivecontrol in ELISA.

1.6 Screening Assay

An ELISA was used for screening of IgG in cell culture supernatants. 96well flat-bottom polystyrene microtiter plates (Greiner, Cat. No 655061)were coated with 50 jil/well PD-1Fc antigen (5 μg/ml) in 0.5 Mcarbonate/bicarbonate buffer, pH 9.6. After incubation overnight in amoist chamber at 4° C. the plates were washed with tris-buffered saline(TBS, 50 mM Tris, pH 7.8, 500 mM sodium chloride) containing 0.01%Triton X-100 (washing buffer) and blocked with 200 μl/well 2% FCS in TBS(blocking buffer) for 1 hour at room temperature (RT) on a shaker. Thewells were washed with washing buffer and 100 μl cell culturesupernatant was added in the appropriate well. Cell culture supernatantfrom SP 2/0 myeloma cells was used as a negative control. As positivecontrol cell culture supernatant from spleen cell culture was used. Theplates were incubated on a shaker for 1 h at RT, followed by severalwashes. For detection of bound antibodies plates were incubated with 50l/well goat anti-mouse IgG (Fab specific) conjugated to alkalinephosphatase (1:5000) in blocking buffer for 1 h at RT on a shaker,followed by several washes and addition of 150 l/well substrate buffer(2 mM 4-nitrophenyl phosphate in 5% diethanolamine+0.5 mM MgCl₂, pH9.8). The optical density (OD) was estimated in a 12-channel Dynex OpsysMR microplate reader at 405 nm. Wells with OD405 nm 2-fold higher thanthe OD405 nm of the average plate value were selected as positive.

1.7 Selection of Stable Antibody Producers

Cells from positive IgG producing cultures were transferred into wellsof a 48-well plate and cultivated for several days (depending on thegrowth characteristics of the cells). An ELISA on PD-1Fc and withoutprecoated antigen in order to select the specific binders was carriedout. The cells from ELISA-positive wells were frozen in freezing medium(90% FCS, 10% DMSO). An aliquot of the cells was further cultivated forproduction of cell culture supernatants for further characterization.

1.8 Limiting Dilution Cloning

As soon as positive wells were identified, hybridoma cells were clonedto reduce the risk of overgrowth by non-producing cells (first cloning).To ensure that the antibodies are truly monoclonal the hybridomas werecloned again (second cloning). The method of limiting dilution was usedfor both cloning procedures. IgG producing cells were distributed intoone 96 well plate containing feeder cells at a density of 1-3 cells perwell. After 8-10 days (depending on growth characteristics) all plateswere visually inspected under the microscope for detection of monoclonalgrowth. Culture supernatants from such wells were screened for specificimmunoglobulin content using the above-described screening assay. Theappropriate clones concerning growth characteristic and ELISA signalwere selected, transferred into wells of a 24-well plate and cultivatedfor some days. A screening assay was performed. This procedure wasrepeated two to three times. The appropriate subclone was selectedrespectively for the second cloning procedure or cultivation forcryopreservation. This procedure resulted in the production of threeanti-PD-1 antibodies: Clone 2, Clone 10 and Clone 19. Clone 2 ischaracterized only with respect to its epitope and binding off-rate.

Example 2 Characterization of the Clone 10 and Clone 19 Antibodies 2.1Reagents Used for Characterization of the Properties of the Antibodies

The following directly labelled antibodies were used: donkey anti-mouseIgG Alexa647 conjugate (Molecular Probes), anti-human CD4 Alexa647conjugate (Serotec Ltd) and anti-human CD4 FITC conjugate (Serotec Ltd).OX7 (mIgG₁ culture supernatant; in-house) and MOPC21 (mIgG₁;Sigma-Aldrich Ltd) were used as isotype controls. Isotype-specificPE-labelled goat anti-mouse IgG₁ and IgG₂a antibodies (STAR81PE andSTAR82PE respectively) were obtained from Serotec Ltd and exhibited <1%cross reactivity with other murine Ig subclasses. Propidium iodide andrabbit IgG were from Sigma Ltd. Clone 19 anti-PD-1 antibody produced asdescribed above was conjugated to Alexa647 using a kit following themanufacturer's instructions (Molecular Probes). IL-2 levels in cellculture supernatants were quantified using the DuoSet Human IL-2 ELISAKit (R&D Systems Ltd).

2.2 Preparation and Isotyping of Antibodies

Hybridoma supernatant was prepared and diluted into sterile, azide-freePBS. Purified stocks of monoclonal antibodies were isotyped at 1 μg/mlin PBS using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (SantaCruz; sc-24958). The isotypes of Clone 19, Clone 10 and Clone 2 wereIgG_(1K).

2.3 Epitope Mapping

Constructs encoding the human extracellular region of PD-1 with thetransmembrane and intracellular regions of murine CD28 were cloned intothe bi-cistronic mammalian expression vector pGFP2-n2 from BioSignalPackard Ltd, which also encodes GFP. Mutant constructs varying by oneamino acid were prepared using the “drastic” mutagenesis approach (Daviset al. Proc Natl Acad Sci USA. 95, 5490-4 (1998)). Plasmids (2 μg/well)were transfected into HEK-293T cells in 6 well plates using Genejuicetransfection reagent (Novagen; 6 μl/well). Mock and no-transfectioncontrols were included with each experiment. Cells were harvested at18-24 hours and stained with anti-PD1 antibodies or isotype controls at10 μg/ml in PBS-azide for 1 h at 4° C. Cells were washed with PBS-Azide,pelleted at 1500 rpm/5 min and primary antibodies were labelled withAlexa647-conjugated donkey anti-mouse IgG (5 μg/ml) in PBS-Azide for 30min at 4° C. Cells were washed as above and resuspended in 200 μlPBS-Azide before being analysed at the flow cytometer. Propidium iodide(5 μg/ml) was added immediately prior to analysis to identify deadcells. GFP-positive (transfected) viable cells were gated and analysedfor binding of anti-PD1 antibodies. Mutants were defined as ‘knock-out’(reducing the percentage of cells bound by the anti-PD1 antibody) or‘knock-down’ (reducing the intensity of antibody staining relative toother PD-1 antibodies).

Following transfection, cells analysed at the flow cytometer were 85-90%viable by propidium iodide exclusion. An example of the binding analysisis shown in FIG. 1. Transfection efficiencies ranged from 15-50% (GFP⁺).Isotype controls were negative on all transfectants. Analysis of thepercentage of GFP⁺ cells that are also positive for Alexa647 (anti-PD-1antibody binding) shows that the L16R and R118D mutations completelyeliminate Clone 19 binding (FIG. 2). All R118D expressing cells bindClone 10, indicating functional expression of PD-1, but have the lowestintensity of all mutants (FIG. 2), suggesting a low level of expression.V18R partially eliminates Clone 19 binding. Clone 10 binds all themutants but for mutants N41K and L103E the binding intensity for thisantibody versus the other PD-1 antibodies is significantly decreased(FIG. 2). The binding analyses thus define two distinct epitopes eachdefined in turn by at least two residues: anti-PD-1 antibody Clone 10binds to a membrane-distal epitope that overlaps with the ligand-bindingregion (Zhang et al. Immunity 20, 337-47 (2004)); Clone 19 binds to amembrane-proximal epitope. The binding-disrupting residues are mappedonto the murine PD-1 crystal structure in FIG. 3.

Example 3 Analysis of Clone 10- and Clone 19-Induced Signaling by aDimeric Form of PD-1 with an Activating Cytoplasmic Domain

To directly compare the signal-generating activities of the antibodies,a dimeric form of PD-1 was generated that consisted of the extracellular(antibody-binding) region of human PD-1 spliced to the transmembraneregion of CD3ζ (to produce dimers) and the cytoplasmic region of CD28(in order to have an “active” readout consisting of IL-2 secretion; FIG.4A).

3.1 Construction of a Dimeric Form of PD-1 for Detecting Anti-PD1Antibody-Induced Activating Signaling in a T-Cell Hybridoma

The hPD-1/mCD3ζWT/mCD28 construct was created in a series of five steps.In step 1, oligonucleotide 1 (left arrow; sequence5′-TAGTAGAGATCTCTCAAGCAGGCCA CCATGCAAATCCCACAGGCGCCGTGG-3′, SEQ ID NO:33), which encodes a BglII restriction site and the rat ribosome bindingsite followed by the initiating codon and the first 21 bases of thesignal peptide-encoding sequence of human PD-1, was used in a polymerasechain reaction (PCR1) with the complement of oligonucleotide 2(5′-TCAGCCGGATCC TTCCAAACCCTGGTGCTCTGCTACTTGCTAGATGG-3′, SEQ ID NO: 34).

Oligonucleotide 2 encodes the last nine residues of the human PD-1extracellular domain (up to residue 170 of the mature polypeptide)inserting a Bam H1 site, followed by 20 bases encoding the NH₂-terminalend of the mouse CD3ζ transmembrane domain. PCR reactions were carriedout under standard conditions. In step 2, oligonucleotide 2 was used ina PCR reaction (PCR2) with the complement of oligonucleotide 3(5′-ATCACAGCCCTGTACCTGAATAGTAGAAGGAATAGACTC-3′, SEQ ID NO: 35) whichencodes the COOH-terminal end of the transmembrane region of mouse CD3c,followed by the first 21 bases encoding the NH₂-terminal end of themouse CD28 cytoplasmic domain. In step 3, the PCR1 and PCR2 reactionproducts were purified, annealed, extended and then amplified in thepresence of oligonucleotide 1 and the complement of oligonucleotide 3,to generate a cDNA encoding the extracellular region of PD-1 fused withthe transmembrane region of CD3c. In step 4, oligonucleotide 3 was usedin a PCR reaction (PCR3) with oligonucleotide 4(5′-CTCGAGCTACTAGGGGCGGTACGCTGCAAA-3′, SEQ ID NO: 36), which encodes theCOOH-terminal end of the cytoplasmic domain of mouse CD28 followed by astop codon and a Xho I restriction site. In step 5, the purified PCR3product was fused with the purified PCR product from step 3 by annealingthe two products, extending the annealed hybrid, and then amplifying itwith oligonucleotides 1 and 4.

Human PD-1 and mouse CD28 cDNA was amplified using pENTRhPD-1/mCD28 astemplate, which was originally constructed from IMAGE clones obtainedfrom Geneservices Ltd (Cambridge UK). Mouse CD3ζ was amplified fromDO11.10 mouse T cell hybridoma cDNA. The fusion PCR products were clonedinto pCR4®-TOPO® (Invitrogen) and the final products sequenced by thedideoxy method. The constructs were cut with BglII and XhoI and insertedinto the lentiviral vector pHR-SIN-BX-IRES-Em.

3.2 Detection of Activating Signaling by the hPD-1/mCD3ζWT/mCD28 Chimera

HEK 293T cells were transfected with pHR-SIN-BX-IRES-Em encodinghPD-1/mCD3ζWT/mCD28, and the supernatant used to infect DO11.10 T-cellhybridomas. Infected DO11.10 cells were propagated and FACS sorted formouse PD-1 and EGFP expression, and then tested for agonistic signalingby the anti-PD-1 antibodies using IL-2 release as a stimulation assayreadout. The IL-2 secretion results indicate that both antibodies arecapable of inducing signaling via the hPD-1/mCD3ζWT/mCD28 chimera;however Clone 19, which binds PD-1 closest to the membrane induces thelargest amount of IL-2 release (representative data is shown in FIG.4B). This supports the notion that the topology of the complex formed bythe antibodies is what determines the relative levels of signalinginduced by agonists. The data also suggest that the degree of agonisticsignaling can be varied with choice of antibody.

Example 4 Analysis of Inhibitory Signaling by Clone 10 and Clone 19Antibodies in Human Peripheral Lymphocytes (PBL)

The antibodies were tested for their ability to inhibit TCR-derivedactivating signals by covalently coupling the antibodies, along withanti-CD3 antibodies, to tosyl-activated DYNALBEADS. The beads were thenadded to cultures of PBL labelled with carboxyfluorescein succinimidylester (CFSE). Proliferation levels were indicated by the fraction ofcells with diluted CFSE determined by flow cytometric analysis.

4.1 Loading and Quantification of Antibody on DYNALBEADS

Tosyl-activated 4.5 μm DYNALBEADS (M450; Invitrogen) were washed in 0.1Msterile phosphate buffer (pH 8) and loaded with 2.5 μg total antibodyper 3×10⁷ beads at 37° C. for 18-24 h with continuous inversion mixing.Rabbit IgG (Sigma) was used to equalise the amount of total antibody perbead-loading reaction. Beads were blocked for at least 30 min in RPMIwith 10% FCS at room temperature and washed three times in serum-freeRPMI. For some experiments, bead-bound antibody was quantified induplicate with saturating amounts of isotype-specific PE-labelled goatantibodies and compared with Quantibrite™ prelabelled quantification kit(BD Biosciences Ltd.). The geometric mean fluorescence PE intensities ofbead singlets (minus background of unloaded beads as a control) wereused to calculate the absolute amount of antibody loaded per bead fromthe standard curve. An example of such a titration is given in FIG. 5.Loaded beads were stored at 4° C. During bead loading the amounts ofanti-CD3 antibody added were varied so that, at the time of theexperiments, the effects of matched sets of beads with near-equivalentlevels of anti-CD3 antibody could be compared. The level of stimulationprovided by anti-CD3 loaded beads was defined as low (resulting in 15%proliferation of bulk lymphocytes at day 5), medium-low (30%proliferation), medium-high (60% proliferation) and high (80%proliferation).

4.2 Proliferation Studies

Fresh heparinized blood was diluted 1:1 with PBS and the lymphocytesisolated by density gradient separation (Ficoll Hypaque). In someexperiments, accessory cells were depleted by plastic adherence for 2 hat 37° C. or with specific antibody-labelled DYNALBEADS (againstCD14/19/8/56). Cells were washed in PBS and RPMI and resuspended at 10⁷cells/ml in serum-free RPMI. Cells were labeled with 25 μM CFSE in PBSfor 10 min in the dark at RT. CFSE was quenched with an equal volume ofFCS at RT for 5 min. Cells were washed 3-5 times with RPMI andresuspended at 10⁶ cells/ml in RPMI+10% FCS+PSG+2-ME (finalconcentration 5×10⁻⁵ M). Antibodies (beads), mitogen or media was addedto relevant wells in 96-well round-bottomed plates and 10⁵ cells/wellwere distributed and incubated at 37° C. for 3-5 days. For proliferationstudies, cells were stained with directly-labelled cell-surfaceantibodies for 1 h at 4° C. Cells were washed with PBS-Azide, pelletedat 1500 rpm/5 min and resuspended in 200 μl PBS-Azide. Cells wereanalysed for CFSE and antibody labelling at the flow cytometer usingFlowJo Flow Cytometry Analysis Software.

4.3 Effects of the Antibodies

In the experimental results described in FIG. 6, the tosyl-activatedbeads used had been incubated with 2.5 μg of total antibody containingup to 2375 ng of anti-PD-1 Clone 10 or Clone 19 antibody, and enoughanti-CD3 antibody to induce ˜25% proliferation in the absence ofanti-PD-1 antibody. Proliferation was measured by CFSE dilution at day5. In experiment 1, visual inspection indicates that inhibition ofproliferation is seen with all three antibodies, with Clones 2 and 19now giving the highest levels of inhibition. For the data that can beanalyzed using automated analysis software (FlowJo), the amount ofinhibition of proliferation by Clone 19 is of the order of 80%. Inexperiment 2, although the degree of proliferation in the presence ofOKT3 only is somewhat reduced (to ˜15%), it is clear that Clones 2 and19 are profoundly inhibiting proliferation; at most the cells that startproliferating undergo one or two rounds of proliferation only. Clone 10is without any inhibitory effect in experiment 2.

Clone 10 antibodies were further tested for their ability to inhibitTCR-derived activating signals by covalently coupling the antibodies,along with anti-CD3 antibodies, to tosyl-activated DYNALBEADS. The beadswere then added to cultures of human CD4⁺ T cells and proliferationmeasured by ³H-thymidine incorporation.

Tosyl-activated 4.5 μm DYNALBEADS (M450; Invitrogen) were washed in 0.1Msterile phosphate buffer (pH 7.5) and loaded with 2 μg of anti-human CD3(clone OKT3) per 1×10⁷ beads at 37° C. for 8 h with continuous inversionmixing. Beads were then washed to remove un-conjugated anti-CD3.Aliquots of the anti-CD3 conjugated beads were then secondarily coatedwith 3 μg of anti-PD-1 antibody or control per 1×10⁷ beads at 37° C. for19 h with continuous inversion mixing. Beads were washed and thenincubated in 0.2M Tris/0.1% BSA (pH 8.5) for 3 hours to inactivate freetosyl groups, followed by washing and re-suspension of beads in PBS/0.1%BSA/2 mM EDTA (pH 7.4). Equal anti-CD3 and antibody coating of the beadsets was confirmed by staining the beads with fluorochrome-labelledisotype-specific antibodies and analysing by flow cytometry.

Fresh heparinized blood was diluted 1:1 with RPMI and the lymphocytesisolated by density gradient separation (Ficoll Hypaque). CD4⁺ T cellswere purified from the whole PBLs by negative selection using MACS (CD4⁺T cell isolation Kit II; Miltenyi Biotec). 1×10⁵ human CD4⁺ T cells/wellwere cultured at a 1:1 ratio with the coated beads in 96-wellround-bottomed plates and incubated at 37° C. for 6 days. Proliferationwas measured at day 6 by addition of 0.5 μCi/well ³H-thymidine for thelast 6 hours of culture. Cells were harvested onto glass-fibre filtersand incorporated ³H-thymidine was measured by β-scintillation counting.

The results in FIG. 13 show the day 6 proliferative response by humanCD4⁺ T cells measured in the presence of anti-CD3 plus Clone 10 antibodyor control coated beads. The data are expressed as percentage of themaximal response (anti-CD3 plus BSA control) and are the mean of 4different donor responses. CD4⁺ T cell proliferation was inhibited inthe presence of Clone 10, so that the average proliferation observed wasonly 37.7% of the maximum.

Clone 19 generally induces stronger signaling by the hPD-1/mCD3ζWT/mCD28chimera than Clone 10 (FIG. 4B) but in some experiments it gives weakerinhibitory signaling by native PD-1 (see, e.g. FIG. 8). It is possiblethat this is because, in some experiments, Clone 19 but not Clone 10ligation results in the phosphorylation of both the ITIM (inhibitory,blue) and the ITSM (activating, red) tyrosine-based signaling motifs ofPD-1 (see FIG. 9).

Example 5 Using Two Antibodies with Non-Overlapping Epitopes to EnhanceSignaling by a Monomeric Receptor

Individual anti-PD-1 antibodies working alone, e.g. Clone 10, arealready inhibitory but it should be possible to significantly enhancethese effects by using pairs of anti-PD-1 antibodies. Initialcharacterization of the signaling properties of the antibodies relied onan assay in which PD-1 was expressed in the form of thehPD-1/mCD3ζWT/mCD28 chimera, which forms a homodimer. This was done inorder to facilitate comparisons with anti-CD28 superagonisticantibodies, since CD28 is also a homodimer. A question that arises is:To what extent is signaling by the hPD-1/mCD3ζWT/mCD28 chimera dependenton its bivalency, and resulting cross-linking? To test this, amonomeric, monovalent form of PD-1, hPD-1/mCD28, that consisted of theextracellular (antibody-binding) and transmembrane regions of human PD-1spliced to the cytoplasmic region of CD28 (in order to have an “active”readout consisting of IL-2 secretion; FIG. 10A), was generated.

5.1 Construction of a Monomeric Form of PD-1 for Detecting Anti-PD1Antibody-Induced Activating Signaling in a T-Cell Hybridoma

The hPD-1/mCD28 construct was created in a series of three steps. Instep 1, oligonucleotide 1 (left arrow; sequence5′-TAGTAGAGATCTCTCAAGCAGGCCACCAT GCAAATCCCACAGGCGCCGTGG-3′, SEQ ID NO:33), which encodes a BglII restriction site and the rat ribosome bindingsite followed by the initiating codon and the first 24 bases of thesignal peptide-encoding sequence of human PD-1, was used in a polymerasechain reaction (PCR1) with the complement of oligonucleotide 2(5′-GCCCAGCCGGCCAGTTCC AAACCTTTTGGGTGCTGGTGGTGGTTGGT-3′, SEQ ID NO: 37).Oligonucleotide 2 encodes the last 23 bases of the human PD-1extracellular domain (up to residue 149 of the mature polypeptide),followed by 24 bases encoding the NH₂-terminal sequence of the mouseCD28 transmembrane region. PCR reactions were carried out under standardconditions. In step 2, oligonucleotide 2 was used in a PCR reaction(PCR2) with the complement of oligonucleotide 3(5′-TTTGCAGCGTACCGCCCCACGCGTTAGTAGCTCGAG-3′, SEQ ID NO: 38) whichencodes the COOH-terminal end of the cytoplasmic domain of mouse CD28, aMlu I restriction site followed by a stop codon and a Xho I restrictionsite. In step 3, the purified PCR2 product was fused with the purifiedPCR1 product from step 1 by annealing the two products, extending theannealed hybrid, and then amplifying it with oligonucleotides 1 and 3.

Mouse CD28 sequence was amplified using pCR4®-TOPO®rCD28/mCD28 astemplate, which was originally amplified from DO11.10 mouse T cellhybridoma cDNA. The human extracellular PD-1 was amplified frompE14hPD-1Long, a gift from Dr Chao Yu of the MRC Human Immunology Unit,Oxford. The fusion PCR products were cloned into pCR4®-TOPO®(Invitrogen)and the final products sequenced by the dideoxy method. The constructswere cut with BglII and XhoI and inserted into the lentiviral vectorpHR-SIN-BX-IRES-Em for infection of DO11.10 cells. Activation of theDO11.10 cells expressing the hPD-1/mCD28 chimera by anti-PD-1 antibodieswas examined using IL-2 secretion as a read-out.

5.2 Lack of Signaling by hPD-1/mCD28 Suggests that Agonistic SignalingMay be Enhanced by Cross-Linking a Monomeric Receptor with TwoAntibodies that Bind to Non-Overlapping Epitopes

Clone 10 and Clone 19 were not agonistic for a chimeric form of humanPD-1, i.e. hPD-1/mCD28, consisting of the monomeric extracellular regionof PD-1 attached to the transmembrane and intracellular signalingdomains of CD28 (FIG. 10), in contrast to the equivalent CD28 construct(containing the homodimeric extracellular domain of rat CD28). Thelikeliest explanation for this is that, because PD-1 is monomeric andCD28 is a homodimer, the attachment of bivalent antibody leads to theassembly of a multimeric array of “cross-linked” CD28 molecules and avery high density of signaling domains (FIG. 11A), whereas the bindingof Clone 10 or Clone 19 brings together only pairs of PD-1 molecules(FIG. 11B). In vivo, therefore, antibodies binding to homodimericreceptors will generally produce stronger signaling than an antibodythat binds to a monomeric receptor. In the case of PD-1, if a multimericassembly of PD-1 molecules could be generated this would be predicted tolead to much more potent signaling (FIG. 11C). The positions of theepitopes of Clone 10 (or Clone 2) and of Clone 19 on opposite “sides” ofPD-1 (FIG. 3) imply that the two antibodies are likely to bind tonon-overlapping surfaces, i.e. that each native PD-1 monomer would becapable of binding both antibodies. This suggests that pairs of theantibodies could be used in vivo to “cross-link” native PD-1 monomers asshown in FIG. 11C. The high-density arrays of sequestered PD-1 moleculesthus generated are expected to produce more potent signaling than wouldbe possible using single antibodies.

5.3 Agonistic Signalling is Enhanced by Cross-Linking a MonomericReceptor with Two Antibodies that Bind to Non-Overlapping Epitopes

To test the idea that pairs of antibodies could be used to “cross-link”native PD-1 monomers and induce enhanced agonistic signaling, DO11.10cells expressing the hPD-1/mCD28 chimeric protein were used in a Clone10/Clone 19 antibody stimulation assay as follows. 96-well flat-bottomedplates (Costar EIA/RIA plates) were coated overnight at 4° C. with 500μg/ml donkey anti-mouse IgG (Jackson Immunoresearch) in coating buffer(15 mM Na2CO3, 35 mM NaHCO3, pH 9.6. Prior to the addition of cells, theplates were washed three times with 200 μl chilled PBS. 5×10⁵ cells werecentrifuged at 1200 rpm for 3 minutes and resuspended in 100 μl completemedium containing the Clone 19 antibody at various concentrations for 30minutes. The cells then washed and subjected to an additional 30 minuteincubation with Clone 10 at various concentrations, before the cellswere plated out in triplicate onto the donkey anti-mouse IgG pre-coated96 well plates. Cells were incubated at 37° C., in 5% CO2 for 48 hoursbefore the cell culture supernatant was removed and assayed for mouseinterleukin-2 (IL-2) by ELISA.

The results of this experiment (FIG. 7) show that at the highestconcentrations, i.e. 100 μg/ml, neither Clone 10 nor Clone 19 initiatesignalling (IL-2 production) in DO11.10 cells expressing the hPD-1/mCD28chimeric protein. However, successive incubations of the antibodies at10-100 μg/ml induced significant levels of IL-2 production. Thissuggests that cross-linking pairs of antibodies could be used to induceenhanced signalling in vivo.

Sequence information for Clones 2, 10 and 19 antibodies Clone 2VK DNA (SEQ ID NO: 1)gacattgtgctgacacagtctcctgcttctttagctgtatctctggggcagagggccaccatctcatgcagggccagcaaaagtgtcagtacatctggctttaattatatacactggtaccaacagaaaccaggacagccacccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggacgaggatgctgcaacctattactgtcagcacagtagggagcttccgctcacgttcggtgctgggaccaagctggaaataaaaVK protein (SEQ ID NO: 2)DIVLTQSPASLAVSLGQRATISCRASKSVSTSGFNYIHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEDEDAATYYCQHSRELPLTFGAGTKLEIKVH DNA (original cloned) (SEQ ID NO: 3)caggtccaactgcagcagcctggggctgaactggtgaagcctggggcttcagtgaagttgtcctgcaaggcttctggctacaccttcaccacctactatttgtactgggtgaggcagaggcctggacaaggccttgagtggattggggggattaatcctagcaatggtggtactaacttcaatgagaagttcaagagcaaggccacactgactgtagacaaatcctccagcacagcctacatgcaactcaacagcctgacatctgaggactctgcggtctattactgtacaagacgggactataggtacgacagaggctttgactactggggccaaggcacctcagtcacagtcVH DNA (mutated to remove splice site) (SEQ ID NO: 5)caggtccaactgcagcagcctggggctgaactggtgaagcctggggcttcagtgaagttgtcctgcaaggcttctggctacaccttcaccacctactatttgtactgggtgaggcagaggcctggacaaggccttgagtggattggggggattaatcctagcaatggtggtactaacttcaatgagaagttcaagagcaaggccacactgactgtagacaaatcctcctctacagcctacatgcaactcaacagcctgacatctgaggactctgcggtctattactgtacaagacgggactataggtacgacagaggctttgactactggggccaaggcacctcagtcacagtcVH protein (SEQ ID NO: 4 or SEQ ID NO: 6)QVQLQQPGAELVKPGASVKLSCKASGYTFTTYYLYWVRQRPGQGLEWIGGINPSNGGTNFNEKFKSKATLTVDKSSSTAYMQLNSLTSEDSAVYYCTRRDYRYDRGFDYWG QGTSVTV Clone 10VK DNA (SEQ ID NO: 7)gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctggtcagaacattgtacatagtaatggaaacacctatttagaatggtacctacagaaaccaggccagtctccaaagctcctgatctacaaagtctccaaccgatifittggggtcccagacaggatcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctttcaaggttcacatgttccattcacgttcggctcggggacaaagctggaaataaaaVK protein (SEQ ID NO: 8)DVLMTQTPLSLPVSLGDQASISCRSGQNIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFFGVPDRISGSGSGTDFTLKISRVEAEDLGVYFCFQGSHVPFTFGSGTKLEIKVH DNA (SEQ ID NO: 9)gatgtgcagcttcaggagtcgggacctggcctggtgaaaccttctcagtctctgtccctcacctgcactgtcactggctactcaatcaccagtgattatgcctggaactggatccggcagtttccaggaaacaaactggagtggatgggctacataaactacagtggtagcactagctacaacccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagatggatcggtagtagcgcctggtacttcgatgtctggggcgcagggaccacggtcacagtcVH protein (SEQ ID NO: 10)DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYINYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARWIGSSAWYFDVWGAGT TVTV Clone 19VK DNA (SEQ ID NO: 11)gaaaatgtgctcacccagtctccagcaatcatgtctgcatctccaggggaaaaggtcaccatgacctgcagggccagctcaagtgtaatttccagttacttgcactggtaccagcagaagtcaggtgcctcccccaaactctggatttatagcacttccaacttggcttctggagtccctgatcgatcagtggcagtgggtctgggacctcttactctctcacaatcagcagtgtggaggctgaagatgctgccacttattactgccagcagtacaatggttacccgctcacgttcggtgctgggaccaagctggaaataaaaVK protein (SEQ ID NO: 12)ENVLTQSPAIMSASPGEKVTMTCRASSSVISSYLHWYQQKSGASPKLWIYSTSNLASGVPDRFSGSGSGTSYSLTISSVEAEDAATYYCQQYNGYPLTFGAGTKLEIKVH DNA (SEQ ID NO: 13)caggttcagctacagcagtctggggctgagctggtgaagcctggggcctcagtgaagatgtcctgcaaggcttttggctacaccttcactacctatccaatagagtggatgaagcagaatcatgggaagagcctagagtggattggaaattttcatccttacaatgatgatactaagtacaatgaaaaattcaagggcaaggccaaattgactgtagaaaaatcctctaccacagtctacttggagctcagccgattaacatctgacgactctgctgtttattactgtgcaagggagaactacggtagtcacgggggttttgtttactggggccaagggactctggtcaccgtcVH protein (SEQ ID NO: 14)QVQLQQSGAELVKPGASVKMSCKAFGYTFTTYPIEWMKQNHGKSLEWIGNFHPYNDDTKYNEKFKGKAKLTVEKSSTTVYLELSRLTSDDSAVYYCARENYGSHGGFVYW GQGTLVTV (CDRsindicated by underlining in amino acid sequences)

TABLE 1 CDR SEQUENCES FOR CLONES 2, 10 AND 19 ANTIBODIES CDR1 CDR2 CDR3Clone 2 RASKSVSTSGFNYIH LASNLES QHSRELPLT Vκ (SEQ ID NO: 15)(SEQ ID NO: 16) (SEQ ID NO: 17) Clone 2 GYTFTTYYLY GINPSNGGTNFNEKFRDYRYDRG VH (SEQ ID NO: 18) KS FDY (SEQ ID NO: 19) (SEQ ID NO: 20)Clone 10 RSGQNIVHSNG KVSNRFF FQGSHVPFT Vκ NTYLE (SEQ ID NO: 22) (SEQ ID(SEQ ID NO: 21) NO: 23) Clone 10 GYSITSDYAWN YINYSGSTSYNPSL WIGSSAWY VH(SEQ ID NO: 24) KS FDV (SEQ ID NO: 25) (SEQ ID NO: 26) Clone 19RASSSVISSYLH STSNLAS QQYNGYPLT Vκ (SEQ ID NO: 27) (SEQ ID NO: 28)(SEQ ID NO: 29) Clone 19 GYTFTTYPIE NFHPYNDDTKYNEKF ENYGSHGG VH(SEQ ID NO: 30) KG FVY (SEQ ID NO: 31) (SEQ ID NO: 32)In Table 1 and the sequences provided above, the heavy chain CDR1s forclones 2, 10 and 19 have been identified according to both the combinedKabat/Chothia numbering system and the Kabat numbering system. All otherCDRs have been identified according to the Kabat numbering system (Kabatet al., 1987, “In sequences of proteins of immunological interest”, U.S.Dept. Health and Human Services, NIH USA. Heavy chain CDRls for clones2, 10 and 19, as identified by the Kabat numbering system, areidentified (underlined amino acids) in Table 1.

We claim:
 1. A monoclonal antibody produced by the hybridoma clone
 2. 2.A monoclonal antibody or antigen binding fragment thereof that binds tothe same epitope as the monoclonal antibody produced by the hybridomaclone
 2. 3. A bispecific, humanized, single-chain, chimeric, syntheticor recombinant antibody that binds to the same epitope as the antibodyproduced by the hybridoma clone
 2. 4. A pharmaceutical compositioncomprising an antibody according to claim
 2. 5. A method of isolatingPD-1 or PD-1-expressing cells comprising contacting an antibodyaccording to claim 2 and isolating said cell.
 6. The method according toclaim 5, wherein the method comprises contacting an additional PD-1specific monoclonal antibody with PD-1 or a PD-1 expressing cell.
 7. Amethod of inducing tolerance to a specific antigen comprisingadministering a specific antigen to a subject in combination with ananti-PD-1 specific antibody having the binding specificity of theantibody according to claim
 2. 8. A method for reducing immune responsesmediated by activated lymphocytes in a subject comprising theadministration of an anti-PD-1 specific antibody having the bindingspecificity of the antibody according to claim 2 to a subject.
 9. Amethod of treating allergies, rheumatoid arthritis, type I diabetesmellitus, multiple sclerosis, inflammatory bowel disease, Crohn'sdisease, systemic lupus erythematosus, tissue, skin and organ transplantrejection or graft-versus-host disease (GVHD) comprising theadministration of an anti-PD-1 specific antibody having the bindingspecificity of the antibody according to claim 2.